The invention relates to cancer and viral vaccines.
The basic rationale for immune therapy against tumors is the induction of an effective immune response against tumor-associated antigens (TAA), which in turn results in immune-mediated destruction of proliferating tumor cells expressing these antigens. For an immune response to be effective against TAAs comprising protein, these antigens must first be endocytosed by macrophages. Within macrophages, TAAs are degraded in the lysosomal compartment and the resulting peptides are expressed on the surface of the macrophage cell membrane in association with MHC Class II molecules. This expression mediates recognition by specific CD4+ helper T cells and subsequent activation of these cells to effect the immune response (Stevenson, 1991, FASEB J. 5:2250; Lanzavecchia, 1993, Science 260:937; Pardoll, 1993, Immunol. Today 14:310). The majority of human TAA molecules have not been defined in molecular terms and as a consequence, no generic version of these molecules is available for exploitation as a potential anti-tumor vaccine. Attempts to modify autologous tumor cells in each individual patient as a means of eliciting an immune response to TAAs have met with little success. In order for autologous TAA-expressing tumor cells to function as effective vaccines, they must be opsonized in some manner so that their phagocytosis by macrophages is enhanced.
To date, available viral vaccines comprise either infectious, attenuated live virus, non-infectious killed virus or subunits thereof. While live attenuated virus is a more efficacious vaccine than killed virus, vaccines of this type are often genetically unstable and thus, have the potential to revert to a wild type, virulent phenotype. In addition, administration of live attenuated vaccines is contraindicated in immunocompromised individuals and in pregnant women. Non-infectious, killed virus vaccines and vaccines comprising viral subunits also have disadvantages in that they have insufficient immunogenic properties and must be administered in high concentrations at frequent intervals.
Anti-Gal, a naturally occurring antibody present in all humans, specifically interacts with the carbohydrate epitope Galxcex11-3Galxcex21-4GlcNAc-R (xcex1-galactosyl epitope). This antibody does not interact with any other known carbohydrate epitope produced by mammalian cells (Galili, 1993, Springer Seminar Immunopathology 15:153). Anti-Gal constitutes approximately 1% of circulating IgG (Galili et al., 1984, J. Exp. Med. 160:1519) and is also found in the form of IgA and IgM (Davine et al., 1987, Kidney Int. 31:1132; Sandrin et al., 1993, Proc. Natl. Acad. Sci. USA 90:11391). It is produced by 1% of circulating B lymphocytes (Galili et al., 1993, Blood 82:2485).
Both anti-Gal and xcex1-galactosyl epitope exhibit a unique distribution pattern in mammals. Anti-Gal is produced abundantly in humans, apes and Old World monkeys, but is absent in New World monkeys, prosimians and non-primate mammals (Galili et al., 1987, Proc. Natl. Acad. Sci. USA 84:1369). In contrast, the xcex1-galacyosyl epitope is found as part of the terminal carbohydrate structure on glycolipids and on carbohydrate chains of glycoproteins, in particular on asparagine (N)-linked carbohydrate chains, in non-primate mammals, prosimians and New World monkeys, but is absent in Old World monkeys, apes and humans (Galili et al., 1987, Proc. Natl. Acad. Sci. USA 84:1369; Galili et al., 1988, J. Biol. Chem. 263:17755).
Asparagine (N)-linked carbohydrate chains similar to those present on mammalian cells, are found attached to envelope glycoproteins of viruses which bud from vertebrate cells (Klenk, 1990, p. 25-37. In: Regenmortel and Neurath (ed.). Immunochemistry of Viruses, II. The Basis for Serodiagnosis and Vaccines. Elsevier Publishers B.V., N.Y.). These carbohydrate chains most often are of the simple high-mannose type containing mannose and N-acetylglucosamine only or of the complex type wherein they contain additional carbohydrates such as galactose, N-acetylglucosamine, fucose and sialic acid. Oligosaccharide chains on viral glycoproteins are synthesized in large part by host cell glycosylation enzymes (Kornfeld et al., 1985, Ann. Rev. Biochem. 54:631; Schlesinger et al., 1986, p. 121-148. In: Schlesinger and Schlesinger (ed.), The Togaviridae and Flaviviridae, Plenum Press, N.Y.; Rademacher et al., 1988, Ann. Rev. Biochem. 57:785)).
Sialylated N-linked complex carbohydrate chains are common on viral glycoproteins and on cell surface glycoproteins since sialyltransferases, which are prevalent in the Golgi apparatus of all mammalian cells, xe2x80x9ccapxe2x80x9d N-acetyllactosaminyl residues of viral carbohydrate chains with sialic acid (Klenk et al., 1980, Curr. Topics Microbiol. Immunol. 90:19-48; Kornfeld et al., 1985, Ann. Rev. Biochem,. 54:631; Rademacher et al., 1988, Ann. Rev. Biochem. 57:785). Geyer et al. (1984, Biochemistry 23:5628) report xcex1-galactosyl epitopes as a major carbohydrate structure on Friend murine leukemia virus.
The invention encompasses compositions and methods for enhancing an immune response to an antigen in an anti-Gal synthesizing animal. In order to elicit an effective, protective immune response to a vaccine, the vaccinating antigen must be processed in vivo by antigen-presenting cells or macrophages. Processing includes uptake of antigen by macrophages, fragmentation of antigen within the lysosomal compartment of these cells and ultimately, presentation of the fragmented peptide on the membrane of the macrophage in association with class II major histocompatibility (MHC) molecules. According to the methods and compositions of the invention, an immune response to an antigen is enhanced by positioning xcex1-galactosyl epitopes either on or juxtaposed to the antigen. When an antigen so treated is administered to an anti-Gal synthesizing animal, opsonization of the antigen is effected by binding of anti-Gal to the xcex1-galactosyl epitopes thereby enhancing phagocytosis and subsequent processing of the antigen by macrophages.
In one aspect, the invention features a method of inducing an immune response to an antigen in an anti-Gal synthesizing animal comprising providing an xcex1-galactosyl epitope containing complex, which complex includes the antigen and a lipid bilayer, and administering an immunizing effective amount of the complex to the animal.
In another aspect, the invention features a method of opsonizing an antigen in an anti-gal synthesizing animal comprising providing an xcex1-galactosyl epitope containing complex, which complex includes the antigen and a lipid bilayer, and administering an immunizing effective amount of the complex to the animal.
In both aspects, the antigen is preferably positioned so that it is juxtaposed to the lipid bilayer. More preferably, the antigen is either inserted in or encapsulated by the lipid bilayer.
In yet another aspect, the invention features a method of opsonizing an antigen in an anti-Gal synthesizing animal comprising encapsulating the antigen in a lipid bilayer to form a complex, incubating the complex in the presence of xcex1-galactosyl epitope containing glycosphingolipids to effect insertion of the xcex1-galactosyl epitopes into the lipid bilayer, and administering the complex so incubated to the animal.
In another aspect, the invention features a method of opsonizing a tumor cell in an anti-Gal synthesizing animal having a tumor comprising obtaining the tumor cell from the animal, incubating the cell in the presence of a preparation of xcex1-galactosyl containing glycosphingolipids to effect insertion of the xcex1-galactosyl epitopes into the membrane of the cell, irradiating the cell so treated and then administering the cell to the animal.
In yet another aspect, the invention features a method of opsonizing a tumor associated antigen in an anti-Gal synthesizing animal having a tumor comprising obtaining a tumor associated antigen containing cell from the animal, extracting the cell membrane of the cell, incubating the cell membrane in the presence of xcex1-galactosyl epitope containing glycosphingolipids to effect insertion of the xcex1-galactosyl epitopes into the membrane and administering the cell membrane so treated to the animal.
The invention includes yet another method of opsonizing a tumor associated antigen in an anti-Gal synthesizing animal having a tumor comprising obtaining a tumor associated antigen containing cell from the animal, incubating the cell in the presence of neuraminidase, further incubating the cell in the presence of xcex11,3 galactosyltransferase and UDP-galactose to effect addition of xcex1-galactosyl epitopes to the tumor associated antigens, irradiating the cell so incubated and administering the cell to the animal.
In yet another aspect, the invention includes a method of opsonizing a tumor associated antigen in an anti-Gal synthesizing animal having a tumor comprising obtaining a tumor associated antigen containing cell from the animal, extracting the cell membrane from the cell, incubating the cell membrane in the presence of neuraminidase, further incubating the cell membrane in the presence of xcex11,3 galactosyltransferase and UDP-galactose to effect addition of xcex1-galactosyl epitopes to the tumor associated antigen and administering the cell membrane so incubated to the animal.
Also included in the invention is an xcex1-galactosyl epitope containing tumor cell of an anti-Gal synthesizing animal.
The invention further includes a method of opsonizing a glycoprotein containing virus in an anti-Gal synthesizing animal comprising propagating the virus in an xcex1-galactosyl epitope containing cell to effect addition of xcex1-galactosyl epitopes to the virus, separating the virus so propagated from the cell, suspending the separated virus in pharmaceutically acceptable carrier and administering the virus to the animal.
In yet another aspect, the invention includes a method of opsonizing a glycoprotein containing virus in an anti-Gal synthesizing animal, wherein the glycoprotein has at its carbohydrate terminus a sialic acid residue. The method comprises incubating the virus in the presence of neuraminidase, further incubating the virus in the presence of xcex11,3 galactosyltransferase and UDP-galactose to effect addition of xcex1-galactosyl epitopes to the glycoprotein, purifying the virus so incubated, suspending it in a pharmaceutically acceptable carrier, and administering the virus to the animal.
In a further aspect, the invention includes a method of opsonizing a non-sialic acid containing glycoprotein containing virus in an anti-Gal synthesizing animal comprising incubating the virus in the presence of xcex11,3 galactosyltransferase and UDP-galactose to effect addition of xcex1-galactosyl epitopes to the glycoprotein, purifying the virus so incubated, suspending the virus in a pharmaceutically acceptable carrier and administering the virus to the animal.
In yet another aspect, the invention features a viral vaccine effective in an anti-Gal synthesizing animal comprising an immunizing effective amount of an xcex1-galactosyl epitope containing virus, or a viral subunit, suspended in a pharmaceutically acceptable carrier.
In preferred embodiments, the tumor is a leukemia, lymphoma, myeloma, melanoma, ovarian carcinoma, lung carcinoma, mammary carcinoma, thyroid carcinoma, colon carcinoma or sarcoma. More preferably, the tumor is leukemia.
In yet other preferred embodiments, the animal is a bird, an Old World monkey or a human, and more preferably, the animal is a human.
In yet more preferred embodiments, the antigen is a component of an infectious agent belonging to the group consisting of a virus, a bacterium, a parasite and a yeast. More preferably, the virus is a member of the following virus families: Orthomyxoviruses, Rhabdoviruses, Hepadnaviruses, Togaviruses, Bunyaviruses, Retroviruses, Paramyxoviruses, Flaviviruses, Herpesviruses, Arenaviruses, or Reoviruses. Even more preferably, the virus is an influenza virus, a rabies virus, a human immunodeficiency virus, a hepatitis B virus, an eastern, western or Venezuelan equine encephalitis virus, a Japanese encephalitis virus, a tick-borne encephalitis virus, a Russian spring-summer encephalitis virus or a Rift Valley fever virus. Most preferably, the virus is influenza virus or eastern equine encephalitis virus and the antigen is influenza virus hemagglutinin or eastern equine encephalitis virus E1 or E2 glycoprotein, or glycoproteins of human immunodeficiency virus.
The invention should also be construed to include other carbohydrates capable of binding to anti-Gal to effect opsonization of an antigen according to the methods of the invention. For example, melibiose and like carbohydrates may bind anti-Gal to effect opsonization. The invention further includes synthetic xcex1-galactosyl epitopes and like carbohydrate structures and also includes synthetic or naturally occurring glycolipids other than GSLs which contain xcex1-galactosyl epitopes capable of interacting with anti-Gal.
By antigen is meant any antigen whose presence in an animal is associated with a disease state, including but not limited to, tumor associated antigens and viral, bacterial, parasitic and fungal antigens. Also included in the term virus are whole infectious organisms or subunits thereof.
By subunit is meant a portion of a virus or antigen which is itself antigenic, i.e., capable of inducing an immune response in an animal. The term should be construed to include subunits which are obtained by both recombinant and biochemical methods.
By tumor cell is meant a cell which is a component of a tumor in an animal, or a cell which is determined to be destined to become a component of a tumor, i.e., a cell which is a component of a precancerous lesion in an animal.
The term tumor associated antigen (TAA) includes all antigens associated with a tumor in an animal, including the traditionally named TAAs.
By opsonization of an antigen is meant binding of the antigen by anti-Gal thereby effecting enhanced phagocytosis and subsequent MHC-associated expression of the antigen by macrophages in the presence of xcex1-galactosyl epitopes compared with phagocytosis and expression of the antigen by macrophages in the absence of xcex1-galactosyl epitopes.
By anti-Gal synthesizing animal is meant an animal which naturally expresses IgG, IgA or IgM anti-Gal antibody.
The term animal should be construed to include all anti-Gal synthesizing animals including those which are not yet known to synthesize anti-Gal. For example, some animals such as those of the avian species, are known not to synthesize xcex1-galactosyl epitopes. Due to the unique reciprocal relationship among animals which synthesize either anti-Gal or xcex1-galactosyl epitopes, it is believed that many animals heretofore untested in which xcex1-galactosyl epitopes are absent may prove to be anti-Gal synthesizing animals. The invention also encompasses these animals.
By immunizing effective amount is meant an amount of antigen which elicits an immune response when administered to an anti-Gal synthesizing animal.
By juxtaposed to is meant that the antigen and xcex1-galactosyl epitope are positioned near each other such that upon binding to anti-Gal they are together phagocytosed by a single macrophage. This term should also be construed to include (i) encapsulation of antigen by a lipid bilayer, (ii) micelle-like structures comprising lipid bilayer and antigen, wherein either the lipid bilayer or antigen has attached thereto xcex1-galactosyl epitopes, and (iii) whole cells or membrane fragments.